1. Field of the Invention
The invention embraces peptide variants based on the domains having inhibitory activity of human bikunin (that is to say the component having inhibitory activity of inter-.alpha.-trypsin inhibitor, or the acid-stable serum trypsin inhibitor, or the urinary trypsin inhibitor), process for preparing the described peptide variants by methods of genetic manipulation using microorganisms (bacteria, lower eukaryotes), as well as medicaments containing these peptide variants. The peptide variants are characterized by their ability to inhibit serine proteases, for example, pancreatic and granulocytic elastase, cathepsin G or plasma kallikrein.
2. Description of Related Art
When proteases reach the extracellular space they are normally rapidly trapped by potent endogenous proteinase inhibitors such as .alpha..sub.1 -proteinase inhibitor (Travis & Salvesen, Ann. Rev. Biochem., 52, 655, 1983). In certain situations this protective mechanism may not operate or at least not operate adequately, and the consequence may be serious pathological states such as the development of emphysema, septic shock, shock lung, ARDS, rheumatoid arthritis, coagulation disorders, kidney and liver failure, inter alia. Proteinase inhibitors with a specific action are of special interest in this connection as potential therapeutics. The proteinase inhibitors which are of particular interest for use in humans have amino acid sequences similar to natural human inhibitors. This particularly applies to long-term therapies such as the treatment of .alpha..sub.1 -proteinase inhibitor deficiencies (development of emphysema) to prevent toxic or allergic side effects.
Although .alpha..sub.1 -proteinase inhibitor is the natural antagonist of neutrophilic elastase, whose extracellular inhibition is the primary aim in an inflammatory event, it is not optimally suited for therapeutic use for several reasons. Its relatively high molecular weight of 53,000 d would require the use of unphysiologically large amounts by weight of the inhibitor. Although the required amounts could be obtained by genetic engineering means, it would be necessary to use even larger amounts of the inhibitor because of the reduced biological half-life in the circulation of a recombinant protein which is not glycosylated in the physiological manner (Matheson et al., J. Biol. Chem., 261, 10404, 1986). In any event, the inhibitor is regarded as susceptible to proteolysis and oxidation.
Both properties further reduce the concentration of the active species. Only the susceptibility to oxidation could be eliminated by genetic engineering means by replacing the methionine residue in the reactive center of the inhibitor (P.sub.1 position) by, for example, leucine (McCourtney et al., Nature, 313, 149, 1985).
The aim of the present invention is to develop proteinase inhibitors, for example, against human leukocyte elastase, cathepsin G or plasma kallikrein having a distinctly lower molecular mass for the treatment of human diseases. In this connection, the aim was to examine whether it is possible to tailor proteinase inhibitors to have a desired inhibitory spectrum by modification of currently known human inhibitors. Conceivable as suitable basic molecules for this would be the subunit having inhibitory activity of human inter-.alpha.-trypsin inhibitor ITI (Schreitmuller et al., Biol. Chem. Hoppe-Seyler, 368, 963, 1987; Gebhard et al., Biol. Chem. Hoppe-Seyler, 369S, 19, 1988; Gebhard et al., FEBS Lett., 229, 63, 1988; Gebhard et al.; Euro. J. Biochem., in press), which is called bikunin hereinafter (FIG. 1), as well as the Kunitz-type trypsin inhibitors of identical structure in the serum (STI) and the urine (UTI), if it were possible by replacing a few amino acid residues to achieve a specific alteration of the natural inhibitory spectrum of these inhibitors (FIG. 1). They are the result of the proteolytic maturation of the primary translation product of a single gene (Kaumeyer et al., Nucleic Acids Res. 14, 7839, 1986) and consist of a N-terminal peptide which can be eliminated with trypsin (amino acid residues 1-21) and two consecutive, structurally related domains (domain 1 is the N-terminal domain, defined as amino acid residues 22-77, and domain 2 is the C-terminal part of the protein, defined as amino acid residues 78-147), and these can also be obtained singly by treatment with trypsin. Both domains have proteinase-inhibitory activity of differing inhibitory specificity (Gebhard & Hochstra.beta.er in: Proteinase Inhibitors, Barrett & Salvesen, eds., Elsevier, 1986, p 375).
Depending on the particular physiological situation, the acute-phase protein bikunin may be present both in complexed form (in ITI or in a complex with immunoglobulins) and in non-complexed form, which is then detectable as STI. UTI is STI which has undergone renal filtration. Accordingly, the individual inhibitors differ only in that they are either associated with other proteins or not, or are detected in different body fluids.
The specific and potent inhibitors of, for example, neutrophilic elastase, cathepsin G or plasma kallikrein based on bikunin domains could be obtained by replacing the amino acid residues in position PI of the reactive centers of domain 1 or domain II of the inhibitor. Furthermore, additional replacements in the domains, for example, in the particular P.sub.2 ' position, are able to improve the inhibitory properties additionally.